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. Author manuscript; available in PMC: 2017 Mar 15.
Published in final edited form as: J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Feb 6;1015-1016:62–73. doi: 10.1016/j.jchromb.2016.02.008

Determination of American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Jin-Yi Wan a,b,c, Chong-Zhi Wang c, Zhi Liu c, Qi-Hui Zhang c, Mark W Musch d, Marc Bissonnette d, Eugene B Chang d, Ping Li b, Lian-Wen Qi b,c,*, Chun-Su Yuan c,*
PMCID: PMC4789086  NIHMSID: NIHMS761347  PMID: 26896573

Abstract

American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 16 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 μg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites.

Keywords: American ginseng, Ginsenosides, Enteric, Microbiota, Ginsenoside Rb1, Compound K, LC-Q-TOF-MS

1. Introduction

American ginseng or Panax quinquefolius is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be active constituents of this botanical [1]. Until now, nearly a hundred ginsenosides have been isolated and characterized from American ginseng, and many of them have shown pharmacological activities [2, 3]. Yet, compared with studies of other ginseng species, such as Panax ginseng and Panax notoginseng, analytical studies of American ginseng and its components are relatively limited [4, 5].

The chemical and pharmacological diversity of different ginseng constituents has been investigated. In studies of many parent compounds of ginseng, attention also has been given to their potential structure-activity relationship [6–8]. Reductionist methodology in ginseng research during in vitro screening has been applied primarily to the bioactivity of parent compounds [9, 10]. However, the bioavailability of ginseng compounds, an important consideration for their effects in vivo, has been overlooked.

Of the commercially available American ginseng products, nearly all are ingested orally. After oral intake, trillions of gut microbiota in the gastrointestinal tract may affect ginseng biotransformation [11]. Metabolic profiles of ginseng by enteric microbiota have been reported in animals [12, 13], and the actions of selected biotransformed metabolites have been elucidated [14]. For example, compound K, a major metabolite in the protopanaxadiols group, has a better bioactivity than its parent compound, ginsenoside Rb1 [15]. In an in vitro study we observed that human enteric microbiota metabolized ginseng parent compounds into 25 metabolites [16]. However, to date, the determination of ginseng metabolites in human biological samples has largely not been carried out. To link the health benefits of ginseng compounds to their effects, we sought to determine the profiles of ginseng and its metabolites after oral administration.

Based on our previous studies, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) can be effectively used to characterize ginseng saponins and their metabolites [16]. This technique provides advanced structural information with high sensitivity, specificity, and versatility in characterizing complex metabolite profiles in matrix-based samples. With this method, all ginseng constituents, even at low concentrations, can be successfully detected.

In a previous study in humans, we analyzed a ginsenoside and its metabolism in plasma after a single oral dose of American ginseng using UPLC-TOF-MS [17]. It was indicated that the metabolite profile might not have been adequately revealed after a single dose regimen in the qualitative study. For the study reported here, we recruited six healthy volunteers to ingest American ginseng for 7 successive days. The 2 g daily ginseng used in this study was within the commonly used therapeutic dose range. Plasma, urine and feces samples were collected at the end of the 7 days and analyzed by LC-Q-TOF-MS. Special attention was paid to the determination of a major ginseng parent compound, ginsenoside Rb1, and its predominant metabolite, compound K.

2. Experimental

2.1. Chemicals and reagents

Reference ginsenosides, including ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, F1, F2, pseudoginsenoside F11, gypenoside XVII, 20S-Rg2, 20R-Rg2, 20S-Rg3, 20R-Rg3, 20S-Rh1, 20R-Rh1, 20S-Rh2, 20R-Rh2, compound K, and protopanaxatriol were purchased from Jilin University (Changchun, China). Ginsenoside Rk3, Rh4, Rk1 and Rg5 were prepared in our laboratory by steaming transformation. The structures are shown in Fig. 1(C), and were elucidated by 1H NMR, 13C NMR and MS. The internal standard (IS) digoxin was obtained from Sigma-Aldrich (St. Louis, USA). The purities of the reference compounds were more than 95%, as determined by HPLC-DAD. Acetonitrile (ACN) and formic acid of HPLC grade were obtained from Merck (Darmstadt, Germany). Deionized water (18 MΩ cm−1) was supplied with a Milli-Q water system (Millipore, Milford, MA, USA). Other reagents were of analytical grade.

Figure 1.

Figure 1

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Ginsenosides detected by LC-Q-TOF-MS in the negative ion mode. (A) Total ion chromatogram (TIC) of ginseng saponin standards; (B) TIC of American ginseng root extract; (C) Chemical structures of 45 American ginseng saponins and their metabolites reported in this study.

2.2. Plant materials

American ginseng roots (P. quinquefolius L.) were obtained from Roland Ginseng, LLC (Wausau, WI, USA). The voucher samples were authenticated by Dr. Chun-Su Yuan and deposited at the Tang Center for Herbal Medicine Research at the University of Chicago (Chicago, IL, USA). Air-dried American ginseng was pulverized to powder and passed through a 40 mesh screen to prepare the sample.

2.3. Human subjects and study protocol

With approval from the Institutional Review Board, six healthy male volunteers (ages 18–45 years) were enrolled in the clinical trial. The subjects were screened for drug abuse disorders or medical contraindications for participation in the study. The volunteers underwent a physical examination and completed a health history questionnaire. On day 1, each subject was asked to fast overnight, and their biological samples (blood, urine and feces) were collected as the control. Then all volunteers were instructed to ingest 1 g of American ginseng powder with water twice a day for 7 consecutive days. On day 7, biological samples from these subjects were again obtained for analysis. The blood samples in the heparin tubes were centrifuged at 4000 rpm for 5 min and the plasma was transferred to Eppendorf tubes. All samples were then immediately chilled on ice and stored at −80°C until analysis.

2.4. Sample preparation

To prevent matrix effects and to concentrate the analytes, all the biological samples were pretreated with solid phase extraction (SPE) before LC-Q-TOF-MS. Oasis HLB columns (1 mL, 30 mg, Waters, Milford, MA, USA) were preconditioned with 2 mL of methanol and equilibrated with 2 mL of deionized water.

The plasma samples (200 μL) were mixed with 20 μL of 2% acetic acid and vortexed for 30 sec at room temperature. Acetic acid was used to release the protein binding ginsenosides and metabolites to increase the recovery of the ingredients for the measurement. The samples were subsequently diluted with 1 mL of physiological saline. The homogenates were loaded onto the preconditioned SPE columns, washed with 2 mL of deionized water and slowly eluted using 1 mL of methanol. The elution was evaporated to dryness under nitrogen at 20°C, and the residue was dissolved in 100 μL of methanol and centrifuged at 13,000 rpm for 10 min before further analysis.

The urine samples were centrifuged at 4,000 rpm for 10 min at room temperature and the supernatants (200 μL) were purified by the preconditioned SPE columns. The cartridge was cleaned with 2 mL of deionized water, and slowly eluted using 1 mL of methanol. The elution was evaporated to dryness under nitrogen at 20°C, and the residue was dissolved in 100 μL of methanol and centrifuged at 13,000 rpm for 10 min.

The feces samples (0.5 g) were extracted with 3 mL of 80% methanol by ultrasound (30°C, 100 Hz) for 15 min. They were then centrifuged at 4,000 rpm for 10 min at room temperature, and the supernatants (200 μL) were purified by the preconditioned SPE columns. The cartridge was cleaned with 2 mL of deionized water and slowly eluted using 1 mL of methanol. The elution was evaporated to dryness under nitrogen at 20°C, and the residue was dissolved in 100 μL of methanol and centrifuged at 13,000 rpm for 10 min.

2.5. LC-MS/MS instrument and conditions

Chromatographic analysis was performed on an Agilent 1290 Series LC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, an online degasser, an auto plate-sampler, and a thermostatically controlled column compartment. Sample separation was carried out at 25°C on an Agilent Zorbax Extend-C18 column (4.6 mm × 250 mm, 5 μm) with a C18 guard column (4.6 mm, 0.2 μm). The mobile phase consisted of water (0.1% formic acid, Solvent A) and acetonitrile (0.1% formic acid, Solvent B), using a gradient elution of 21% B at 0–15 min, 21%–30% B at 15–18 min, 30%–33% B at 18–30 min, 33% B at 30–34 min, 33%–45% B at 34–40 min, 45%–60% B at 40–50 min, 60%–80% B at 50–55 min, 80%–100% B at 55–60 min, and 100% B at 60–65 min. The flow rate was kept at 1 mL/min, and the injection volume of plasma, urine, and feces samples was set at 10 μL, 5 μL, and 10 μL, respectively.

Detection was performed by a 6530 Q-TOF mass spectrometer (Agilent) with a Dual electrospray ionization (ESI) source. The operating parameters were optimized as follows: drying gas (N2) flow rate, 10.0 L/min; drying gas temperature, 320°C; nebulizer, 35 psig; capillary, 3500 V; OCT RFV, 750 V; and fragmentor voltage, 120 V. Mass spectra were recorded across the range m/z 100–3000 in both positive and negative ion modes. The operation and acquisition of data were monitored by Agilent LC-Q-TOF-MS MassHunter Acquisition Software (Version B.04.00). A low flow of calibrant solution A was applied to obtain high accuracy measurements. The calibrant solution A was a reference mass solution of purine and HP-0921, which was used to provide reference ions in the Agilent mass spectrometer. The reference ions applied in different ion modes were distinguishing; 121.0508, 922.0097 (positive ions) and 112.9855, 1033.9881 (negative ions). The dosage of reference mass solution added in the system was adjusted on the basis of the actual MS response value in LC-Q-TOF-MS, and the signal intensities of the reference ions were expected to be higher than 10000. Thus, the calibrant solution A was imported from reference nebulizer, which was set at 5 psi as default.

2.6. Method validation

The method was validated for selectivity, linearity, limits of detection (LODs), lower limits of quantification (LLOQs), and recovery based on FDA Guidance for Industry-Bioanalytical Method Validation [18]. The selectivity was investigated by comparing the chromatograms of six individual blank human plasma with those of corresponding standard plasma samples spiked with the analytes and IS and plasma samples after oral administration of American ginseng. The linearity was evaluated by analyzing calibration standards at each concentration level over three consecutive days. The calibration curves were constructed by plotting the peak area ratio (y) of analytes to IS versus plasma concentrations (x) of analytes using a 1/x2 weighted least-squares linear regression model. LODs were defined as the lowest amount of analytes that could be detected, which was determined as the concentration with a signal to noise ratio (S/N) of 3:1. LLOQs was served as the lowest concentration of the standard curve, giving a S/N of 10:1. The recoveries of Rb1 and compound K were determined in the plasma at three spiking levels (10, 100 and 1000 ng/mL), by comparing the peak areas obtained from samples of spiked matrices and the peak areas obtained from direct injection of known amounts of standard solutions.

2.7. Data analysis

Mass data were analyzed with Agilent MassHunter Workstation software (Version B.05.00). Accurate mass measurements (error <5 ppm) were obtained by comparing the theoretical mass of molecular ions and/or fragment ions to achieve an empirical molecular formula. The levels of parent ginsenosides and metabolites in individual subjects and their mean ± S.D. were calculated.

3. Results and discussion

3.1. Analytical condition optimization

In this study, LC-Q-TOF-MS was used for the online structural characterization of American ginseng saponins and their metabolites in human plasma, urine and feces samples.

Considering the chemical complexity of the biological matrixes, the effects of chromatographic conditions, including the column, mobile phases and modifiers, were evaluated. It was shown that Zorbax Extend-C18 column was the most suitable for the separation of ginsenosides and the relevant metabolites. A solvent system of water/acetonitrile with gradient elution was optimized for better resolution in a shorter time. Moreover, a low concentration of formic acid (0.1%) added in the mobile phase improved the peak symmetry and enhanced the ionization efficiency of major analysts, such as ginsenoside Rb1 and compound K.

In MS analysis both positive and negative ion modes were employed to obtain the abundance information for the structural characterization of complex constituents from the matrix. Due to the presence of formic acid in the mobile phase, the deprotonated molecular ion [M-H]− and typical solvent adduct ion [M+HCOO]− were generated in ESI negative ion mode, providing information on the molecular mass. In particular, [M-H]− (m/z 1107.59) and [M+HCOOH]− (m/z 1153.60) of ginsenoside Rb1, and [M-H]− (m/z 621.44) and [M+HCOOH] (m/z 667.44) of compound K were observed in the negative ion mode of the MS spectrum. [M+H]+ (m/z 1131.59) of ginsenoside Rb1 and [M+H]+ (m/z 645.43) of compound K were detected in the positive ion mode. The negative ion mode gives much lower background noise with less interference, so it was used for the following MS/MS experiments. Meanwhile, the collision energy was adjusted for optimum performance in negative MS/MS, and 50V was ultimately selected.

Under these conditions, 25 reference ginsenosides were well separated. The total ion chromatogram (TIC) of ginseng saponin standards is shown in Fig. 1A in the negative ion mode. Fig. 1B shows the TIC of American ginseng root extract in the negative ion mode. A total of 19 major ginseng saponins were detected and identified, and are listed in accordance with previous publications [19, 20].

3.2. Methodological validation for quantitative analysis

Ginsenoside Rb1 and compound K in the plasma were quantified by LC–MS/MS. There was no endogenous interference at the retention times of the analytes and IS. The two analytes exhibited good linear regression (r2 > 0.995) within the test ranges of 0.005–5 μg/ml. The LODs of Rb1 and compound K were estimated to be 1.55 and 0.95 ng/mL, and the LLOQs were 3.75 and 2.50 ng/mL. These data showed that the method was sensitive for the quantitative evaluation of the analytes. The mean recoveries in plasma at three different concentrations (10, 100 and 1000 ng/mL) for Rb1 were 83.2%, 87.5% and 88.3%, respectively, and for compound K were 85.1%, 89.4% and 89.8%, respectively. These results suggested that this method is acceptable for quantification of ginsenoside Rb1 and compound K in the biological samples.

3.3.Characterization of American ginseng constituents and their metabolites in plasma

After oral administration, American ginseng saponins inevitably come into contact with different kinds of gut microbiota and are transformed before they are absorbed from the gastrointestinal tract. In this study, human plasma, orally administered with American ginseng, was analyzed by the established LC-Q-TOF-MS method. To differentiate the constituent-related peaks from endogenous effects, blank plasma (Fig. 2A) was compared with a typical plasma sample collected 7 days after oral ginseng administration (Fig. 2B) in the analysis.

Figure 2.

Figure 2

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LC-Q-TOF-MS analysis of ginsenoside Rb1 and compound K in human plasma samples. (A) TIC of blank plasma; (B) TIC of a typical plasma sample collected 7 days after oral administration of American ginseng; (C) Extracted ion chromatogram (EIC) of ginsenoside Rb1 of typical plasma sample; (D) EIC of compound K; (E) Signal intensity of ginsenoside Rb1 and compound K in six individual subjects representing their relative abundance measured by peak area with a 0.01 Da mass window; (F) Average intensity of ginsenoside Rb1 and compound K in the six subjects.

A total of 15 peaks were identified as the main components in human plasma, including 10 original ginsenosides and 5 metabolites of American ginseng. Table 1 shows the retention time, experimental and calculated mass m/z, ppm error, and fragment ions in the MS/MS stage of these compounds in the negative ion mode, which were identified by comparing retention times and MS data with those of reference compounds. We observed that 20S-ginsenoside Rg2, 20R-ginsenoside Rg2, ginsenoside F2, 20R-ginsenoside Rg3, and compound K were the major metabolites detected in the plasma of the six subjects. The relative abundance of the compounds measured by signal intensity (peak area) in the extracted ion chromatograms (EICs) is also shown in Table 1. From the data, the concentrations of only 5 detected metabolites in the plasma were relatively low. Since we used sensitive LC-Q-TOF-MS for analysis, some low-level parent ginsenosides were also detected in the present study.

Table 1.

LC-MS/MS data in the negative ion mode of compounds identified in plasma samples after oral American ginseng administration (n = 6).

No. Compound Formula tR
(min)		 Signal
Intensity		 [M-H]− or [M+HCOO]− Fragment ions in the negative mode with the energy 50V CID
m/z Calc m/z Diff
(ppm)
1 Gjnsenoside Rg1 C42H72O14 15.72 1368 845.4911 845.4904 −0.86 475.3775[M-H-glc-glc]−,637.4289[M-H-glc]−,799.4854[M-H]−,845.4911[M+HCOO]−
2 Gjnsenoside Re C48H82O18 16.01 1643 991.5468 991.5483 1.60 475.3762[M-H-glc-rha-glc]−,637.4306[M-H-glc-rha]−,783.4892[M-H-glc]−,945.5429[M-H]−, 991.5468[M+HCOO]−
4 Pseudoginsenoside F11 C42H72O14 24.83 1868 845.4896 845.4904 1.01 491.3747[M-H-rha-glc]−,653.4282[M-H-rha]−,799.4853[M-H]−,845.4896[M+HCOO]−
5 Gjnsenoside Rb1 C54H92O23 26.31 1168 1153.6018 1153.6011 −0.59 459.3818[M-H-glc-glc-glc-glc]−,621.4346[M-H-glc-glc-glc]−,783.4953[M-H-glc-glc]−, 945.5435[M-H-glc]−,1107.5927[M-H]−,1153.6018[M+HCOO]−
7 Ginsenoside Rc C53H90O22 27.97 920 1123.5905 1123.5906 0.07 459.3827[M-H-araf-glc-glc-glc]−,621.4335[M-H-araf-glc-glc]−,783.4846[M-H-araf-glc]−, 945.5422[M-H-araf]−,1077.5819[M-H]−,1123.5905[M+HCOO]−
8 20S-Ginsenoside Rg2 C42H72O13 28.71 1502 829.4942 829.4955 1.65 475.3714[M-H-rha-glc]−,637.4235[M-H-rha]−,783.4887[M-H]−,829.4942[M+HCOO]−
10 20R-Ginsenoside Rg2 C42H72O13 29.47 1347 829.4960 829.4955 −0.52 475.3746[M-H-rha-glc]−,637.4310[M-H-rha]−,783.4896[M-H]−,829.4960[M+HCOO]−
11 Gjnsenoside Rb2 C53H90O22 30.19 830 1123.5921 1123.5906 −1.41 459.3823[M-H-arap-glc-glc-glc]−,621.4476[M-H-arap-glc-glc]−,783.4954[M-H-arap-glc]−, 945.5462[M-H-arap]−,1077.5878[M-H]−,1123.5921[M+HCOO]−
14 Ginsenoside Rb3 C53H90O22 30.83 547 1123.5933 1123.5906 −2.52 459.3845[M-H-xyl-glc-glc-glc]−,621.4252[M-H-xyl-glc-glc]−,783.4842[M-H-xyl-glc]−, 945.5355[M-H-xyl]−,1077.5866[M-H]−,1123.5933[M+HCOO]−
18 Gjnsenoside Rd C48H82O18 34.96 3383 991.5502 991.5483 −1.99 459.3837[M-H-glc-glc-glc]−,621.4441[M-H-glc-glc]−,783.4959[M-H-glc]−,945.5436[M-H]−, 991.5502[M+HCOO]−
21 Gypenoside XVII C48H82O18 38.70 2651 991.5471 991.5483 1.29 459.3858[M-H-glc-glc-glc]−,621.4347[M-H-glc-glc]−,783.4835[M-H-glc]−,945.5414[M-H]−, 991.5471[M+HCOO]−
28 Ginsenoside F2 C42H72O13 42.77 4990 829.4947 829.4955 1.01 459.3864[M-H-glc-glc]−,621.4403[M-H-glc]−,783.4882[M-H]−,829.4947[M+HCOO]−
33 20S-Ginsenoside Rg3 C42H72O13 44.75 3864 829.4935 829.4955 2.54 459.3816[M-H-glc-glc]−,621.4322[M-H-glc]−,783.4874[M-H]−,829.4935[M+HCOO]−
34 20R-Ginsenoside Rg3 C42H72O13 45.06 2771 829.4938 829.4955 2.16 459.3809[M-H-glc-glc]−,621.4373[M-H-glc]−,783.4862[M-H]−,829.4938[M+HCOO]−
42 Compound K C36H62O8 49.69 11402 667.4420 667.4427 1.08 459.3853[M-H-glc]−,621.4362[M-H]−,667.4420[M+HCOO]−
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All listed compounds are confirmed with reference compounds.

Our previous research data showed that compound K is a primary metabolite of American ginseng root [21]. A narrow mass window of 0.01 Da to restructure the EICs of ginsenoside Rb1 and compound K of the typical plasma sample is shown in Fig. 2C and 2D, respectively. The relative abundance of ginsenoside Rb1 and of compound K was measured by the signal intensity in the plasma of six individual subjects for semi-quantitation (Fig. 2E). For ginsenoside Rb1, subject CW had the highest relative abundance and subject MB had the lowest. Meanwhile, for compound K, subject MM had the highest relative abundance and subject CY had the lowest. Fig. 2F shows that the average intensity of ginsenoside Rb1 and compound K in the six subjects was (0.1118 ± 0.0591) × 104 and (1.1069 ± 0.0710) × 104, respectively. The calculated average plasma levels for Rb1 and compound K were 8.6 and 58.4 ng/mL, respectively. In addition, if the plasma level of the major compound, compound K, was set as 100%, the percentage for Rb1 was 14.7%. It was suggested that the concentration of ginsenoside Rb1 decreased after oral ginseng administration and it was transferred to compound K before entering the systemic circulation.

3.4. Characterization of American ginseng constituents and their metabolites in urine

The TIC of a typical urine sample collected at 7 days (Fig. 3B) was analyzed and compared with a blank urine sample (Fig. 3A). We identified 20 peaks as the main components in urine, including 10 original ginsenosides and 10 metabolites.

Figure 3.

Figure 3

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LC-Q-TOF-MS analysis of ginsenoside Rb1 and compound K in human urine samples. (A) TIC of blank urine; (B) TIC of a typical urine sample collected 7 days after oral administration of American ginseng; (C) EIC of ginsenoside Rb1 of typical urine sample; (D) EIC of compound K; (E) Signal intensity of ginsenoside Rb1 and compound K in six individual subjects; (F) Average intensity of ginsenoside Rb1 and compound K in the six subjects.

Table 2 shows the retention time, experimental and calculated mass m/z, ppm error, and fragment ions in the MS/MS stage of these compounds in six urine samples in the negative ion mode. These were identified by comparing retention times and MS data with those of reference compounds. 20S-ginsenoside Rg2, 20R-ginsenoside Rg2, 20S-ginsenoside Rh1, 20R-ginsenoside Rh1, ginsenoside F1, ginsenoside F2, 20R-ginsenoside Rg3, compound K, 20S-ginsenoside Rh2 and 20R-ginsenoside Rh2 were major metabolites detected in urine, more than those found in plasma. The relative abundance of metabolites measured by signal intensity in the EICs are also listed.

Table 2.

LC-MS/MS data in the negative ion mode of compounds identified in urine samples after oral American ginseng administration (n = 6).

No. Compound Formula tR
(min)		 Signal
Intensity		 [M+HCOO]− Fragment ions in the negative mode with the energy 50V CID
m/z Calc m/z Diff
(ppm)
1 Ginsenoside Rg1 C42H72O14 15.77 8971 845.4900 845.4904 0.51 475.3736[M-H-glc-glc]−,637.4374[M-H-glc]−,799.4838[M-H]−,845.4900[M+HCOO]−
2 Ginsenoside Re C48H82O18 16.55 14258 991.5470 991.5483 1.39 475.3765[M-H-glc-rha-glc]−,637.4357[M-H-glc-rha]−,783.4753[M-H-glc]−, 945.5456[M-H]−, 991.5470[M+HCOO]−
4 Pseudoginsenoside F11 C42H72O14 24.93 10046 845.4896 845.4904 1.01 491.3739[M-H-rha-glc]−,653.4251[M-H-rha]−,799.4826[M-H]−,845.4896[M+HCOO]−
5 Ginsenoside Rb1 C54H92O23 26.45 7720 1153.6012 1153.6011 −0.05 459.3826[M-H-glc-glc-glc-glc]−,621.4374[M-H-glc-glc-glc]−,783.4956[M-H-glc-glc]−, 945.5421[M-H-glc]−,1107.5851[M-H]−,1153.6012[M+HCOO]−
7 Ginsenoside Rc C53H90O22 28.15 3103 1123.5878 1123.5906 2.58 459.3842[M-H-araf-glc-glc-glc]−,621.4364[M-H-araf-glc-glc]−,783.4881[M-H-araf-glc]−, 945.5363[M-H-araf]−,1077.5866[M-H]−,1123.5878[M+HCOO]−
8 20S-Ginsenoside Rg2 C42H72O13 28.76 9731 829.4959 829.4955 −0.52 475.3726[M-H-rha-glc]−,637.4272[M-H-rha]−,783.4908[M-H]−,829.4959[M+HCOO]−
9 20S-Ginsenoside Rh1 C36H62O9 29.27 12439 683.4378 683.4376 −0.33 475.3761[M-H-glc]−,637.4329[M-H]−,683.4378[M+HCOO]−
10 20R-Ginsenoside Rg2 C42H72O13 29.55 5628 829.4967 829.4955 −1.54 475.3717[M-H-rha-glc]−,637.4297[M-H-rha]−,783.4880[M-H]−,829.4967[M+HCOO]−
11 Ginsenoside Rb2 C53H90O22 30.24 2746 1123.5892 1123.5906 1.28 459.3834[M-H-araf-glc-glc-glc]−,621.4332[M-H-araf-glc-glc]−,783.4855[M-H-araf-glc]−, 945.5244[M-H-araf]−,1077.5853[M-H]−,1123.5892[M+HCOO]−
13 20R-Ginsenoside Rh1 C36H62O9 30.65 10829 683.4383 683.4376 −1.12 475.3749[M-H-glc]−,637.4308[M-H]−,683.4383[M+HCOO]−
14 Ginsenoside Rb3 C53H90O22 30.96 2118 1123.5883 1123.5906 2.11 459.3853[M-H-araf-glc-glc-glc]−,621.4322[M-H-araf-glc-glc]−,783.4890[M-H-araf-glc]−, 945.5313[M-H-araf]−,1077.5870[M-H]−,1123.5883[M+HCOO]−
16 Ginsenoside F1 C36H62O9 33.97 11073 683.4373 683.4376 0.45 475.3742[M-H-glc]−,637.4314[M-H]−,683.4373[M+HCOO]−
18 Ginsenoside Rd C48H82O18 35.41 11832 991.5474 991.5483 0.97 459.3878[M-H-glc-glc-glc]−,621.4366[M-H-glc-glc]−,783.4907[M-H-glc]−, 945.5487[M-H]−, 991.5474[M+HCOO]−
21 Gypenoside XVII C48H82O18 38.85 7721 991.5475 991.5483 0.86 459.3867[M-H-glc-glc-glc]−,621.4324[M-H-glc-glc]−,783.4837[M-H-glc]−, 945.5479[M-H]−, 991.5475[M+HCOO]−
28 Ginsenoside F2 C42H72O13 42.82 13424 829.4962 829.4955 −0.90 459.3842[M-H-glc-glc]−,621.4383[M-H-glc]−,783.4889[M-H]−,829.4962[M+HCOO]−
33 20S-Ginsenoside Rg3 C42H72O13 44.76 8910 829.4958 829.4955 −0.39 459.3844[M-H-glc-glc]−,621.4374[M-H-glc]−,783.4909[M-H]−,829.4958[M+HCOO]−
34 20R-Ginsenoside Rg3 C42H72O13 45.08 8627 829.4968 829.4955 −1.66 459.3864[M-H-glc-glc]−,621.4336[M-H-glc]−,783.4943[M-H]−,829.4968[M+HCOO]−
42 Compound K C36H62O8 49.77 21352 667.4426 667.4427 0.12 459.3838[M-H-glc]−,621.4374[M-H]−,667.4426[M+HCOO]−
43 20S-Ginsenoside Rh2 C36H62O8 50.83 8236 667.4419 667.4427 1.24 459.3851[M-H-glc]−,621.4348[M-H]−,667.4419[M+HCOO]−
44 20R-Ginsenoside Rh2 C36H62O8 51.14 7150 667.4418 667.4427 1.4 459.3848[M-H-glc]−,621.4342[M-H]−,667.4418[M+HCOO]−
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All listed compounds are confirmed with reference compounds.

The presence of 20S-ginsenoside Rh2 and 20R-ginsenoside Rh2 in urine was discovered for the first time, especially considering that the pair compounds have the same elemental composition and similar MS/MS spectrums, but generate different pathways with compound K. We speculate that 20S-ginsenoside Rh2 and 20R-ginsenoside Rh2 detected in urine may be caused by hepatoenteric circles and other factors.

EICs of ginsenoside Rb1 and compound K in a typical urine sample are shown in Fig. 3C and 3D, respectively. The results of signal intensity in the urine of six individual subjects showed that for ginsenoside Rb1 and compound K, subject MM had the highest relative abundance and subject ZZ had the lowest (Fig. 3E). The average intensity of ginsenoside Rb1 and compound K in the six subjects was (0.7720 ± 0.1245) × 104 and (2.1352 ± 0.1195) × 104, respectively (Fig. 3F), and these values were calculated as 56.8 ± 9.2 and 109.8 ± 6.1 ng/mL, respectively [17, 22].

3.5. Characterization of American ginseng constituents and their metabolites in feces

The TICs of a blank feces sample and a typical feces sample collected at 7 days are compared in Fig. 4A and 4B. Surprisingly, 36 peaks were identified in human feces, including 10 original ginsenosides and 16 metabolites. Table 3 shows the retention time, experimental and calculated mass m/z, ppm error, and fragment ions in the MS/MS stage of these compounds identified in feces samples in the negative ion mode. Some of the compounds were identified with corresponding reference compounds based on their retention times and MS data. Some were assigned based on a comparison of data obtained from previous publications [23, 24].

Figure 4.

Figure 4

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LC-Q-TOF-MS analysis of ginsenoside Rb1 and compound K in human feces samples. (A) TIC of blank feces; (B) TIC of a typical feces sample collected 7 days after oral administration of American ginseng; (C) EIC of ginsenoside Rb1 of typical feces sample; (D) EIC of compound K; (E) Signal intensity of ginsenoside Rb1 and compound K in six individual subjects; (F) Average intensity of ginsenoside Rb1 and compound K in the six subjects.

Table 3.

LC-MS/MS data in the negative ion mode of compounds identified in feces samples after oral American ginseng administration (n = 6).

No. Compound Formula tR
(min)		 Signal
Intensity		 [M-H]− or [M+HCOO]− Fragment ions in the negative mode with the energy 50V CID
m/z Calc m/z Diff
(ppm)
1 Ginsenoside Rg1 a C42H72O14 15.88 15449 845.4900 845.4904 0.51 475.3790[M-H-glc-glc]−,637.4356[M-H-glc]−,799.4848[M-H]−,845.4900[M+HCOO]−
2 Ginsenoside Re a C48H82O18 16.80 14817 991.5504 991.5483 −2.20 475.3779[M-H-glc-rha-glc]−,637.4304[M-H-glc-rha]−,783.4816[M-H-glc]−,945.5442[M-H]−, 991.5504[M+HCOO]−
3 Pseudoginsenoside F11a C42H72O14 24.90 7721 845.4888 845.4904 2.01 491.3763[M-H-rha-glc]−,653.4298[M-H-rha]−,799.4861[M-H]−,845.4888[M+HCOO]−
5 Ginsenoside Rb1 a C54H92O23 26.44 7843 1153.6021 1153.6011 −0.86 459.3829[M-H-glc-glc-glc-glc]−,621.4370[M-H-glc-glc-glc]−,783.4956[M-H-glc-glc]−, 945.5383[M-H-glc]−,1107.5965[M-H]−,1153.6021[M+HCOO]−
7 Ginsenoside Rc a C53H90O22 28.17 11400 1123.5897 1123.5906 0.81 459.3897[M-H-araf-glc-glc-glc]−,621.4403[M-H-araf-glc-glc]−,783.4724[M-H-araf-glc]−, 945.5477[M-H-araf]−,1077.5869[M-H]−,1123.5897[M+HCOO]−
8 20S-Ginsenoside Rg2 a C42H72O13 28.75 15908 829.4957 829.4955 −0.26 475.3783[M-H-rha-glc]−,637.4246[M-H-rha]−,783.4904[M-H]−,829.4957[M+HCOO]−
9 20S-Ginsenoside Rh1 a C36H62O9 29.27 17974 683.4375 683.4376 0.14 475.3728[M-H-glc]−,637.4319[M-H]−,683.4375[M+HCOO]−
10 20R-Ginsenoside Rg2 a C42H72O13 29.47 5564 829.4932 829.4955 2.93 475.3767[M-H-rha-glc]−,637.4303[M-H-rha]−,783.4836[M-H]−,829.4932[M+HCOO]−
11 Ginsenoside Rb2 a C53H90O22 30.33 4529 1123.5880 1123.5906 2.39 459.3847[M-H-arap-glc-glc-glc]−,621.4454[M-H-arap-glc-glc]−,783.4951[M-H-arap-glc]−, 945.5495[M-H-arap]−,1077.5842[M-H]−,1123.5880[M+HCOO]−
130 20R-Ginsenoside Rh1 a C36H62O9 30.56 6334 683.4372 683.4376 0.61 475.3751[M-H-glc]−,637.4318[M-H]−,683.4372[M+HCOO]−
14 Ginsenoside Rb3 a C53H90O22 31.00 11280 1123.5933 1123.5906 −2.52 459.3858[M-H-xyl-glc-glc-glc]−,621.4272[M-H-xyl-glc-glc]−,783.4911[M-H-xyl-glc]−, 945.5347[M-H-xyl]−,1077.5870[M-H]−,1123.5933[M+HCOO]−
16 Ginsenoside F1 a C36H62O9 33.93 6027 683.4387 683.4376 −1.74 475.3799[M-H-glc]−,637.4332[M-H]−,683.4387[M+HCOO]−
18 Ginsenoside Rd a C48H82O18 35.43 22428 991.5476 991.5483 0.76 459.3862[M-H-glc-glc-glc]−,621.4374[M-H-glc-glc]−,783.4892[M-H-glc]−, 945.5422[M-H]−, 991.5476[M+HCOO]−
21 Gypenoside XVII a C48H82O18 38.83 6173 991.5471 991.5483 1.29 459.3844[M-H-glc-glc-glc]−,621.4358[M-H-glc-glc]−,783.4846[M-H-glc]−, 945.5415[M-H]−, 991.5471[M+HCOO]−
23 Compound Mc-1 C47H80O17 40.02 12339 961.5400 961.5378 −2.45 459.3781[M-H-araf-glc-glc]−,621.4344[M-H-araf-glc]−,783.4760[M-H-araf]−,915.5333[M-H]−, 961.5400[M+HCOO]−
25 Compound O C47H80O17 40.44 7322 961.5360 961.5378 1.91 459.3812[M-H-arap-glc-glc]−,621.4434[M-H-arap-glc]−,783.4992[M-H-arap]−,915,5314[M-H]−, 961.5360[M+HCOO]−
26 Gypenoside IX C47H80O17 40.96 7806 961.5373 961.5378 0.50 459.3847[M-H-xylp-glc-glc]−,621.4425[M-H-xylp-glc]−,783.4960[M-H-xylp]−,915.5321[M-H]−,961.5373[M+HCOO]−
27 Ginsenoside Rg6 C42H70O12 42.62 5318 811.4854 811.4849 −0.61 457.3683[M-H-rha-glc]−,619.4237[M-H-rha]−,765.4799[M-H]−,811.4854[M+HCOO]−
28 Ginsenoside F2 a C42H72O13 42.80 28340 829.4965 829.4955 −1.28 459.3863[M-H-glc-glc]−,621.4354[M-H-glc]−,783.4913[M-H]−,829.4965[M+HCOO]−
29 Ginsenoside F4 C42H70O12 43.14 13384 811.4840 811.4849 1.21 457.3713[M-H-rha-glc]−,619.4178[M-H-rha]−,765.4784[M-H]−,811.4840[M+HCOO]−
30 Ginsenoside Rk3 a C36H60O8 43.72 7882 665.4276 665.4270 −0.93 457.3774[M-H-glc]−,619.4224[M-H]−,665.4276[M+HCOO]−
31 Zingibroside R1 C42H66O14 44.26 23676 793.4371 793.4380 1.11 455.3586[M-H-glc-glc-acid −,631.4345[M-H-glc]−,793.4371[M-H]−
322 Ginsenoside Rh4 a C36H60O8 44.31 22888 665.4281 665.4270 −1.74 457.3763[M-H-glc]−,619.4224[M-H]−,665.4281[M+HCOO]−
33 20S-Ginsenoside Rg3 a C42H72O13 44.74 93907 829.4978 829.4955 −2.94 459.3885[M-H-glc-glc]−,621.4336[M-H-glc]−,783.4913[M-H]−,829.4978[M+HCOO]−
34 20R-Ginsenoside Rg3 a C42H72O13 45.08 50189 829.4970 829.4955 −1.92 459.3845[M-H-glc-glc]−,621.4380[M-H-glc]−,783.4909[M-H]−,829.4970[M+HCOO]−
35 Compound Mc C41H70O12 45.95 96372 799.4862 799.4849 −1.68 459.3871[M-H-araf-glc]−,621.4367[M-H-araf]−,753.4807[M-H]−,799.4862[M+HCOO]−
36 Compound Y C41H70O12 46.28 16440 799.4841 799.4849 1.10 459.3852[M-H-arap-glc]−,621.4384[M-H-arap]−,753.4788[M-H]−,799.4841[M+HCOO]−
37 Compound Mx C41H70O12 46.43 43523 799.4875 799.4849 −3.41 459.3850[M-H-xylp-glc]−,621.4544[M-H-xylp]−,753.4818[M-H]−,799.4875[M+HCOO]−
38 Protopanaxatriol a C30H52O4 47.69 200054 521.3852 521.3848 −0.92 475.3798[M-H]−,521.3852[M+HCOO]−
39 Calenduloside E C36H56O9 47.78 16112 631.3847 631.3852 0.72 455.3558[M-H-glc-acid]−,631.3847[M-H]−
40 Ginsenoside Rk1 a C42H70O12 48.75 19950 811.4861 811.4849 −1.53 441.3746[M-H-glc-glc]−,603.4284[M-H-glc]−,765.4808[M-H]−,811.4861[M+HCOO]−
41 Ginsenoside Rg5 a C42H70O12 49.20 54581 811.4862 811.4849 −1.66 441.3782[M-H-glc-glc]−,603.4275[M-H-glc]−,765.4810[M-H]−,811.4862[M+HCOO]−
42 Compound K a C36H62O8 49.77 1964307 667.4429 667.4427 −0.37 459.3841[M-H-glc]−,621.4375[M-H]−,667.4429[M+HCOO]−
43 20S-Ginsenoside Rh2 a C36H62O8 50.79 14763 667.4431 667.4427 −0.69 459.3832[M-H-glc]−,621.4378[M-H]−,667.4431[M+HCOO]−
444 20R-Ginsenoside Rh2 a C36H62O8 51.10 10737 667.4435 667.4427 −1.33 459.3844[M-H-glc]−,621.4381[M-H]−,667.4435[M+HCOO]−
45 Protopanaxadiol C30H52O3 62.57 349343 505.3904 505.3898 −1.20 459.3850[M-H]−,505.3904[M+HCOO]−
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a

Confirmed with reference compounds. Others were tentatively identified based on previous reports [23, 24].

The results indicate that the total number of compounds detected is highest in feces. It is possible that some of these compounds were not absorbed by the gastrointestinal tract or that the absorbed ones had levels below our detection limit. Interestingly, the major metabolites detected in the feces of our six subjects were mostly consistent with those found in our previous in vitro studies of the biotransformation of American ginseng by human intestinal microflora [16]. Specifically, metabolites Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 could be produced by intestinal microflora via dehydration. The relative abundance of the metabolites measured in the EICs is listed in Table 3, which also showed the highest of all of the biological samples.

EICs of ginsenoside Rb1 and compound K in the typical feces sample are shown in Fig. 4C and 4D, respectively. The results of signal intensity in the feces showed that for ginsenoside Rb1 and compound K (Fig. 4E), subject MM had the highest relative abundance and CW had the lowest. The average intensity of ginsenoside Rb1 and compound K in the feces samples was (0.007843 ± 0.003891 ) × 106 and (1.964307 ± 0.187727) × 106, respectively (Fig. 4F). These values were calculated as 57.7 ± 28.6 ng/mL and 10.06 ± 0.96 μg/mL, respectively [17, 22].

Essentially, there was no compound absorption in the colon, and microbiota continuously biotransformed Rb1 into compound K. Thus, it is reasonable to assume that very high compound K levels and low Rb1 levels would be found in feces after the oral administration of American ginseng. Fig. 5 summarizes the biotransformation pathways of protopanaxadiol ginsenosides of American ginseng from parent compounds to metabolites by human enteric microbiota.

Figure 5.

Figure 5

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Biotransformation pathways of protopanaxadiol ginsenosides by human intestinal microbiota.

4. Conclusion

In this study, we tested American ginseng saponins and their metabolites in plasma, urine and feces samples from six human subjects. A total of 5, 10, and 16 metabolites were detected in the plasma, urine and feces, respectively, using LC-Q-TOF-MS technology. Out of all the metabolites, compound K was found at the highest levels in these biological samples. It is likely that ginsenoside Rb1 was biotransformed into compound K via gastrointestinal microbiota. This biotransformation appears to contribute to the reported pharmacological effects of American ginseng and compound K [25, 26]. The high number of metabolites detected in feces from this study is consistent with our previous in vitro human intestinal microflora investigation [16]. In addition, the dehydration process affects the transformation, and metabolites Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 are generated. The ginseng metabolites reported in this study should be further evaluated for their biological effects.

Highlights.

  • American ginseng saponins and their metabolites in human plasma, urine and feces samples after oral administration by LC-Q-TOF-MS

  • The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 16 in feces

  • The levels of ginsenoside Rb1 and compound K were determined, and compound K had remarkably high level in all three kinds of biological samples

  • Ginsenosides Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration in human feces

Acknowledgments

We thank Sally Kozlik for editing the manuscript. This work was supported in part by grants from the NIH/NCCAM AT004418 and AT005362, the Senior Talent Cultivation Program of Jiangsu University (15JDG069), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD).

Footnotes

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